Exosomes are a major type of extracellular vesicle playing crucial roles in cell-cell signaling in the nervous system. Exosomes are also implicated in the spread of disease-linked molecules such as amyloid ? peptide (A?) and the microtubule-associated protein tau in the nervous system, thereby promoting the pathogenesis of Alzheimer?s disease (AD). Two central steps in exosome-mediated cargo transport are exosome secretion from the donor cell and exosome entry into the recipient cell. The machinery mediating these steps, however, remains unknown. The major goal of this collaborative project is to address this fundamental question, capitalizing on our team?s unique and complementary expertise in vesicle transport, exosome biology, genetic screens, and AD pathology. In our preliminary studies, we developed a comprehensive set of two-dimensional (2D) and three- dimensional (3D) neuronal culture systems based on human induced pluripotent stem cells (iPSCs). In addition, we established complementary approaches to isolate and characterize exosomes. Furthermore, we performed a genome-wide CRISPR genetic screen to dissect exosome-mediated cargo transport and identified a group of candidate genes. Here, we will take strategic advantage of these preliminary data to identify the machinery mediating exosome secretion and entry in neurons and glial cells. We will then delineate the molecular mechanisms of the machinery using our unique biochemical reconstitution systems. Finally, we will examine the functional roles of the machinery in the propagation of AD-linked molecules in 2D cultures of neuronal and glial cells and 3D cultures of neurospheroids. Successful completion of this proposed research will fill major gaps in our understanding of neuronal signaling and AD pathogenesis. Knowledge of how exosomes transport cargo will facilitate the development of therapeutic strategies to block exosome-mediated propagation of AD-linked molecules. Such therapeutics are expected to be particularly powerful for preventing and treating sporadic late- onset AD, which intrinsically has a lower probability of producing pathogenic A? and tau species and therefore is expected to be strongly dependent on exosome-carried ?seeds? to generate disease-causing aggregates.